In this proposal, I outline a set of three related yet independent studies of circadian neural output. Recent advances in imaging and data analysis capture information regarding network phenomena with increasing spatial and temporal precision. The circadian pacemaker system we study is advantageous in that it produces physiological activity both spontaneously and rhythmically. In the previous cycle, we used planar illumination methods to perform 24 hr in vivo brain-wide scans of the circadian neural circuit. That work introduced a new concept to theories of how the circadian network encodes time: we showed that the molecularly synchronous pacemaker network displays sequential activation by different identified pacemaker groups across the day. Further we found pacemaker cell interactions, in the form of neuropeptide-mediated delay, represents a key mechanism to effect sequential pacemaker activation. The scientific premise for this project rests on the need to extend those observations on circadian pacemaker neuronal plasticity and to understand how these activity patterns are transmitted to downstream centers. Here I propose work that continues real-time in vivo studies of neuronal activity patterns for the core Drosophila circadian pacemaker neurons. It also continues the focused analysis of neuropeptide modulatory mechanisms that critically regulate the specific timing of pacemaker activity. To extend the scope of our initial studies, and to provide a better understanding of neuronal properties of pacemakers and pacemaking networks, this program will pursue three Aims. Aim 1 will better define daily Ca2+ dynamics in pacemakers by (i) performing in-depth, high frequency sampling, and (ii) by determining the sub-cellular mechanisms underlying these fluctuations. Aim 2 will pursue a Structure-Function analysis of the PDF receptor (PDFR), especially its C-terminus, to understand the regulatory mechanisms that control the quantitative extent of daily PDF signaling. It also seeks to identify key PDFR regulatory proteins. Aim 3 seeks to extend the scope of our work beyond the circadian pacemaker network to identify downstream circuit elements: we will focus on subsets of neurons in the Central Complex for which preliminary evidence suggests an involvement in daily rhythmic physiology associated with locomotion. Jet-lag, shift-work and disturbances in sleep-activity cycles all contribute to degrade mental and physical well-being. Two major causes of death (stroke and cardiac arrest) display clear time- of-day variation, yet, we have little understanding of the causal links between circadian clock functions and disease mechanisms. This research program is dedicated to a better understanding of fundamental circadian output mechanisms.